Project Summary Vibrio cholerae (Vc) is the causative agent of the severe diarrheal disease cholera, a disease that affects 3-5 million people each year. V. cholerae is a native to aquatic ecosystems; its worldwide disruption and epidemic capability makes V. cholerae a significant public health threat. Cholera is acquired by ingestion of Vc-contaminated food or water. Upon entering the gastrointestinal tract, V. cholerae responds to unknown in vivo environmental cues to activate expression of the ToxR regulon, a regulatory cascade that controls expression of genes that facilitate colonization and virulence factor production. Once established in the small intestine, Vc replicates to a high cell density before exiting the host in the diarrheal purge that is the hallmark of disease. This represents a significant dissemination event that contributes to epidemic spread of cholera. Phenotypic analysis has shown that the repression of the ToxR regulon occurs late in infection while genes that are important for dissemination and transmission are activated. The regulatory elements and signals that contribute to late infection phenotypes are unknown. Previous studies in our laboratory characterized the activity of a cyclic dipeptide metabolite, cFP, and showed that cFP inhibits virulence factor production by a ToxR-dependent mechanism. The function of ToxR in the establishment of Vc in the gastrointestinal tract has been well studied; however, the mechanism by which ToxR integrates environmental signals to modulate gene expression remains poorly understood. The finding that ToxR can function to repress virulence expression in response to environmental cues significantly expands the role of ToxR in Vc pathogenesis. This exemplifies the importance of delineating the mechanisms of ToxR signal transduction to gain to a better understanding of virulence regulation. ToxR is a one-component transcriptional activator that contains a cytoplasmic DNA-binding domain and a periplasmic domain proposed to function in signal sensing; these domains are linked by a transmembrane domain. Preliminary studies indicate that the periplasmic domain is required for ToxR-dependent gene induction in response to certain stimuli. Therefore, we hypothesize that ToxR functions to transduce signals by directly interacting with environmental cues via its periplasmic domain. Two aims are proposed to test this hypothesis. Aim 1 will define the role of the ToxR periplasmic domain (PPD) in environmental sensing. This includes characterizing the interaction between effector molecules and the PPD and identifying functional regions within the PPD that contribute to ToxR activity. Aim 2 will characterize the mechanism of ToxR-dependent transcription activation. Results from these studies will provide significant insight into ToxR signal transduction pathways. Furthermore, determining the mechanism of action of antivirulence compounds, like cyclic dipeptides, will contribute to the development of novel approaches to combat cholera.